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CN105209493B - HER3 monoclonal antibody specific for diagnosing and treating purposes - Google Patents

HER3 monoclonal antibody specific for diagnosing and treating purposes Download PDF

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Publication number
CN105209493B
CN105209493B CN201480015195.5A CN201480015195A CN105209493B CN 105209493 B CN105209493 B CN 105209493B CN 201480015195 A CN201480015195 A CN 201480015195A CN 105209493 B CN105209493 B CN 105209493B
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seq
antibody
cdr
her3
structural domain
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CN105209493A (en
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N·张
Z·安
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University of Texas System
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University of Texas System
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Abstract

Provide separation or recombination anti-HER3 monoclonal antibody.In some cases, the antibody of embodiment can be used for the detection of human disease's such as cancer, the treatment of diagnosing and/or treating property.

Description

HER3 monoclonal antibody specific for diagnosing and treating purposes
(it is integrally logical by the U.S. Provisional Patent Application No. 61/782,770 submitted this application claims on March 14th, 2013 Cross and be incorporated herein by reference) equity.
Sequence table is incorporated to
The sequence table for including in the file for being known as " UTFH.P0295WO_ST25.txt " in company with submission is submitted by electronics (it is 25KB (such as in MicrosoftMiddle measurement) and it is created on March 11st, 2014), the sequence List is incorporated herein by reference.
Background of invention
1. invention field
Present invention relates in general to carcinobiology fields.More specifically, it is related to treatment and detection for cancer HER3 targets monoclonal antibody.
2. the description of related fields
EGF-R ELISA (EGFR) family (ErbB/HER) is by 4 known members: EGFR (HER1, erbB- 1), HER2 (erbB-2), HER3 (erbB-3) and HER4 (erbB-4) composition.Each receptor protein is having the same basic Structure is made of extracellular amino terminal domains, single cross-film formation sequence and intracellular cytoplasmic domain.ErbB signal transduction With complicated network, there is the network interaction ligand more than 11 kinds to be used for different binding specificities and signal transduction The activation of approach.The complex contents and interaction of HER receptor and ligand provide great potential for significant signal diversification.
More and more evidences show HER3 in HER target therapeutic agent (including small molecule tyrosine kinase inhibitors (TKI), such as Gefitinib, Tarceva and Lapatinib and HER family receptors target such as bent appropriate list of monoclonal antibody Anti-, Cetuximab, Victibix and handkerchief trastuzumab) resistance mechanism in play an important role.Heregulin/nerve modulation egg White (NRG) is the member with the HER3 and HER4 complicated ligand family to interact.Neuregulin combines activation ErbB3, And lead to formation and the downstream signal of HER3 of heterodimeric receptor compound by PI3K/AKT and Ras/Raf7MAPK approach The activation of conduction.Therefore, the HER3 associativity monoclonal antibody that block nerves regulatory protein combines, which has, blocks HER3 signal to turn Lead and inhibit the potentiality of cancer cell multiplication.
Summary of the invention
Described herein is potent blocking HER3 signal transduction and the HER3 monoclonal antibody for inhibiting cancer cell multiplication. Therefore, in the first embodiment, the separation of specific binding HER3 or recombination monoclonal antibody is provided.Certain In aspect, the antibody with and Rab46, Rab1210, Rab189 or Rab774 monoclonal antibody compete combination to HER3.? In some terms, the antibody may include Rab46, Rab1210, Rab189 or Rab774 monoclonal antibody heavy chain variable region and/ Or all or part of light chain variable region.In other aspects, the antibody may include corresponding to from embodiment of the present invention The light variable chains of Rab46, Rab1210, Rab189 or Rab774 monoclonal antibody and/or the first, second of weight variable chains and/ Or the amino acid sequence of third complementarity-determining region (CDR).
In some aspects, the isolated antibody is comprising with Rab46, Rab1210, Rab189 or Rab774 heavy chain and gently The CDR region of chain amino acid sequence has the CDR sequence of at least 80%, 90% or 95% identity.In other aspects, except one 1 or 2 amino acid substitution, missing on a or multiple CDR or insertion are outer, the antibody include with Rab46, Rab1210, The identical CDR region in the area Rab189 or Rab774CDR.For example, the antibody may include wherein CDR sequence in VH CDR1、VH CDR2、VH CDR3、VL CDR1、VLCDR2 and/or VLRelative to Rab46, Rab1210, Rab189 or Rab774 in CDR3 The CDR of monoclonal antibody includes the CDR of 1 or 2 amino acid substitution.Therefore, in some specific aspects, embodiment of the present invention Antibody include (a) and Rab46 (SEQ ID NO:9), Rab1210 (SEQ ID NO:15), Rab189 (SEQ ID NO:21) Or the V of Rab774 (SEQ ID NO:27)HCDR1 has the first V of at least 80% identityHCDR；(b) with Rab46 (SEQ ID NO:10), Rab1210 (SEQ ID NO:16), Rab189 (SEQ ID NO:22) or Rab774 (SEQ ID NO:28) VHCDR2 has the 2nd V of at least 80% identityHCDR；(c) with Rab46 (SEQ ID NO:11), Rab1210 (SEQ ID NO:17), the V of Rab189 (SEQ ID NO:23) or Rab774 (SEQ ID NO:29)HCDR3 has at least 80% identity The 3rd VHCDR；(d) with Rab46 (SEQ ID NO:12), Rab1210 (SEQ ID NO:18), Rab189 (SEQ ID ) or the V of Rab774 (SEQ ID NO:30) NO:24LCDR1 has the first V of at least 80% identityLCDR；(e) with Rab46 (SEQ ID NO:13), Rab1210 (SEQ ID NO:19), Rab189 (SEQ ID NO:25) or Rab774 (SEQ ID NO:31) VLCDR2 has the 2nd V of at least 80% identityLCDR；(f) with Rab46 (SEQ ID NO:14), The V of Rab1210 (SEQ ID NO:20), Rab189 (SEQ ID NO:26) or Rab774 (SEQ ID NO:32)LCDR3 tool There is the 3rd V of at least 80% identityLCDR.In some aspects, such antibody be comprising human IgG (for example, IgG1, The IgG of IgG2, IgG4 or genetic modification) aforementioned CDR on main chain humanization or go immune antiboidy.
In other aspects, isolated antibody includes that CDR sequence corresponding with monoclonal antibody Rab46 has at least 80% First V of identityH, the 2nd VH, the 3rd VH, the first VL, the 2nd VLWith the 3rd VLCDR sequence, the CDR sequence is respectively by SEQ ID NO:9,10,11,12,13 and 14 are shown.In one aspect, isolated antibody includes the CDR with monoclonal antibody Rab46 The identical CDR sequence of sequence.
On the other hand, isolated antibody include with Rab46 (SEQ ID NO:1), hRab46H-1 (SEQ ID NO: Or the V of hRab46H-2 (SEQ ID NO:34) 33)HStructural domain has the V of at least about 80% identityHStructural domain；With with Rab46 The V of (SEQ ID NO:2), hRab46L-1 (SEQ ID NO:35) or hRab46L-2 (SEQ ID NO:36)LStructural domain has At least about V of 80% identityLStructural domain.For example, the antibody may include the V with hRab46H-1HStructural domain (SEQ ID NO:33) with the V of at least 95% identityHStructural domain and with hRab46L-1 (SEQ ID NO:35) or hRab46L-2 (SEQ ID NO:36) VLStructural domain has the V of at least 95% identityLStructural domain.Therefore, in some respects, antibody include with The V of hRab46H-1HThe identical V of structural domain (SEQ ID NO:33)HStructural domain and V with hRab46L-1LStructural domain (SEQ ID NO:35) identical VLStructural domain.In other aspects, antibody includes the V with hRab46H-1HStructural domain (SEQ ID NO:33) phase Same VHStructural domain and V with hRab46L-2LThe identical V of structural domain (SEQ ID NO:36)LStructural domain.On the other hand, Antibody includes the V with hRab46H-2HStructural domain (SEQ ID NO:34) has the V of at least 95% identityHStructural domain and with The V of hRab46L-2 (SEQ ID NO:36) or hRab46L-1 (SEQ ID NO:35)LStructural domain has at least 95% identity VLStructural domain.For example, antibody may include the V with hRab46H-2HThe identical V of structural domain (SEQ ID NO:34)HStructural domain With the V with hRab46L-2LThe identical V of structural domain (SEQ ID NO:36)LStructural domain, or the V with hRab46H-2HStructural domain (SEQ ID NO:34) identical VHStructural domain and V with hRab46L-1LThe identical V of structural domain (SEQ ID NO:35)LStructure Domain.In specific example, the isolated antibody may include and monoclonal antibody Rab46, HER3-hMab-A9, HER3- The V of hMab-A10, HER3-hMab-A11 or HER3-hMab-A12HAnd VLThe identical V of structural domainHAnd VLStructural domain.Other Aspect, the antibody are HER3-hMab-A9, HER3-hMab-A10, HER3-hMab-A11 or HER3-hMab-A12 antibody.
In other aspects, the isolated antibody includes respectively as shown in SEQ ID NO:15,16,17,18,19 and 20 With the corresponding CDR sequence of monoclonal antibody Rab1210 have at least 80% identity the first VH, the 2nd VH, the 3rd VH, One VL, the 2nd VLWith the 3rd VLCDR sequence.In one aspect, the isolated antibody includes and monoclonal antibody Rab1210 The identical CDR sequence of CDR sequence.
On the other hand, the isolated antibody includes and Rab1210 (SEQ ID NO:3), hRab1210H-1 The V of (SEQ ID NO:37) or hRab1210H-2 (SEQ ID NO:38)HStructural domain has the V of at least about 80% identityHKnot Structure domain；With Rab1210 (SEQ ID NO:4), hRab1210L-1 (SEQ ID NO:39) or hRab1210L-2 (SEQ ID NO: 40) VLStructural domain has the V of at least about 80% identityLStructural domain.Therefore, in some respects, the antibody include with The V of hRab1210H-1HStructural domain (SEQ ID NO:37) has the V of at least 95% identityHStructural domain and and hRab1210L- The V of 1 (SEQ ID NO:39) or hRab1210L-2 (SEQ ID NO:40)LStructural domain has the V of at least 95% identityLKnot Structure domain.Such as the antibody may include the V with hRab1210H-1HThe identical V of structural domain (S EQ ID NO:37)HStructural domain and With the V of hRab1210L-1LThe identical V of structural domain (SEQ ID NO:39)LStructural domain, or the V with hRab1210H-1HStructural domain (SEQ ID NO:37) identical VHStructural domain and V with hRab1210L-2LThe identical V of structural domain (SEQ I D NO:40)LKnot Structure domain.In other aspects, antibody includes the V with hRab1210H- 2HStructural domain (SEQ ID NO:38) is same at least 95% The V of one propertyHStructural domain and V with hRab1210L-2 (SEQ ID NO:40) or hRab1210L-1 (SEQ ID NO:39)LKnot Structure domain has the V of at least 95% identityLStructural domain.For example, the antibody may include the V with hRab1210H-2HStructural domain (SEQ ID NO:38) identical VHStructural domain and V with hRab1210L-2LThe identical V of structural domain (SEQ ID NO:40)LKnot Structure domain, or the V with h Rab1210H-2HThe identical V of structural domain (SEQ ID NO:38)HStructural domain and with hRa b1210L-1's VLThe identical V of structural domain (SEQ ID NO:39)LStructural domain.In specific example, the isolated antibody may include and Dan Ke The V of grand antibody Rab1210, HER3-hMa b-A13, HER3-hMab-A14, HER3-hMab-A15 or HER3-hMab-A16H And VLThe identical V of structural domainHWith VL structural domain.In other aspects, the antibody is HE R3-hMab-A13, HER3-hMab- A14, HER3-hMab-A15 or HER3-hMab- A16 antibody.
In other aspects, the isolated antibody includes and is encoded respectively by SEQ ID NO:21,22,23,24,25 and 26 With the corresponding CDR sequence of monoclonal antibody Rab189 have at least 80% identity the first VH, the 2nd VH, the 3rd VH, first VL, the 2nd VLWith the 3rd VLCDR sequence.In one aspect, the isolated antibody includes the CDR with monoclonal antibody Rab189 The identical CDR sequence of sequence.
On the other hand, the isolated antibody includes the V with Rab189HStructural domain (SEQ ID NO:5) has extremely The V of few about 80% identityHStructural domain and V with Rab189LStructural domain (SEQ ID NO:6) has at least about 80% identity VLStructural domain.In one aspect, the isolated antibody includes the V with monoclonal antibody Rab189HAnd VLStructural domain is identical VHAnd VLStructural domain.
In other aspects, the isolated antibody includes respectively as shown in SEQ ID NO:27,28,29,30,31 and 32 With the corresponding CDR sequence of monoclonal antibody Rab774 have at least 80% identity the first VH, the 2nd VH, the 3rd VH, first VL, the 2nd VLWith the 3rd VLCDR sequence.In one aspect, the isolated antibody includes the CDR with monoclonal antibody Rab774 The identical CDR sequence of sequence.
On the other hand, the isolated antibody includes the V with Rab774HStructural domain (SEQ ID NO:7) has extremely The V of few about 80% identityHStructural domain and V with Rab774LStructural domain (SEQ ID NO:8) has at least about 80% identity VLStructural domain.In one aspect, the isolated antibody includes the V with monoclonal antibody Rab774HAnd VLStructural domain is identical VHAnd VLStructural domain.
In some respects, the antibody of embodiment can be IgG (for example, IgG1, IgG2, IgG3 or IgG4), IgM, IgA, genetically modified IgG isotype or its antigen-binding fragment.The antibody can be Fab', F (ab') 2, F (ab') 3, Monovalent scFv, bivalent scFv, bispecific or single domain antibody.The antibody can be human antibody, humanized antibody or go to be immunized Antibody.In other aspects, the isolated antibody is Rab46, Rab1210, Rab189 or Rab774 antibody.
In some respects, the antibody can be conjugated in preparation, chemotherapeutant, toxin or radionuclide.
In one embodiment, it provides comprising the V containing Rab46HStructural domain CDR1-3 (SEQ ID NO:9, 10 and 11)；The V of Rab1210HThe CDR1-3 (SEQ ID NO:15,16 and 17) of structural domain；The V of Rab189HStructural domain CDR1-3 (SEQ ID NO:21,22 and 23)；The V of Rab774HThe CDR1-3's (SEQ ID NO:27,28 and 29) of structural domain Antibody VHThe recombinant polypeptide of structural domain.In another embodiment, it provides comprising containing Rab46 (SEQ ID NO:12,13 With 14), Rab1210 (SEQ ID NO:18,19 and 20), Rab189 (SEQ ID NO:24,25 and 26) or Rab774 (SEQ ID NO:30,31 and V 32)LThe antibody V of the CDR1-3 of structural domainLThe recombinant polypeptide of structural domain.
In some embodiments, it provides and contains antibody V disclosed herein comprising codingHOr VLThe antibody of structural domain Or the isolated polynucleotide molecule of the nucleic acid sequence of polypeptide.
In other embodiments, the place of the monoclonal antibody or recombinant polypeptide that generate embodiment of the present invention is provided Chief cell.In some respects, host cell is that mammalian cell, yeast cells, bacterial cell, ciliate cells or insect are thin Born of the same parents.In some aspects, host cell is hybridoma.
In other embodiments, the method for manufacturing antibody of the invention is provided, the method includes the tables in cell Up to the V of one or more coding antibody disclosed hereinHOr VLThe polynucleotide molecule of chain and from described in the cell purification resist Body.
In a further embodiment, there is the pharmaceutical composition comprising antibody or antibody fragment discussed herein.This Class composition also includes pharmaceutically acceptable carrier and may include or can not include other active constituent.
In embodiments of the invention, the method for treating the subject for suffering from cancer, the method packet are provided Include a effective amount of antibody disclosed herein of application.In some aspects, the antibody is monoclonal antibody of the invention, such as Rab46、Rab1210、Rab189、Ra b774、HER3-hMab-A9、HER3-hMab-A10、HER3-hMab-A11、HER3 - HMab-A12, HER3-hMab-A13, HER3-hMab-A14, HER3-hMab-A15 or HER3-hMab-A16 antibody or comprising The recombinant polypeptide of antibody section derived from from it.
In some aspects, cancer can be breast cancer, lung cancer, head and neck cancer, prostate cancer, the cancer of the esophagus, tracheocarcinoma, the cancer of the brain, Liver cancer, bladder cancer, gastric cancer, cancer of pancreas, oophoroma, uterine cancer, cervix cancer, carcinoma of testis, colon and rectum carcinoma or cutaneum carcinoma.
It in one aspect, can antibody described in systemic administration.In a further aspect, can it is intravenous, intradermal, tumor is interior, it is intramuscular, In peritonaeum, antibody described in subcutaneous or local application.The method, which may also include to subject, applies at least the second anti-cancer therapies.The The example of two anti-cancer therapies includes but is not limited to operative treatment, chemotherapy, radiotherapy, cold therapy, hormonotherapy, is immunized Therapy or cytokine therapy.
In other aspects, the method, which may also include to subject, applies composition of the invention more than once, such as 1, 2,3,4,5,6,7,8,9,10,15,20 times or more times.
In another embodiment, the method for the cancer for detecting subject is provided, the method includes tests Relative to presence of the raised HER3 in the sample from subject is compareed, wherein the test bag is included sample and this paper Disclosed in antibody contact.For example, the method can be method in vitro or in vivo.
Certain embodiments be related to specifically bind HER3 antibody or comprising specifically bind HER3 separation and/or The antibody of recombination or the recombinant polypeptide composition of polypeptide.In some aspects, the antibody or polypeptide have with it is provided herein The all or part of any monoclonal antibody has, at least have or at most have 80,85,90,95,96,97,98,99 or The sequence of 100% identity (or may originate from any range therein).In other aspects, the separation and/or recombination it is anti- Body or polypeptide have, at least have or at most have 10,11,12,13,14,15,16,17,18,19,20,21,22,23,24, 25、26、27、 28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、 43、44、45、46、47、48、49、 50、51、52、53、54、55、56、57、 58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、 73、74、 75、76、77、78、79、80、81、82、83、84、85、86、87、 88、89、90、91、92、93、94、95、96、97、98、99、 The continuous amino acid of 100 or more any sequences from sequence provided herein or the combination of such sequence.
In other aspects, the antibody or polypeptide of embodiment of the present invention include one or more amino disclosed herein One or more amino acid sections of any amino acid sequence of acid sequence.For example, the antibody or polypeptide may include 1,2,3, 4,5,6,7,8,9,10 or more amino acid sections, the amino acid section include length be about, at least or at most 5,6, 7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、 22、23、24、25、26、27、28、29、30、31、32、 33、34、35、36、 37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、 52、53、54、55、56、57、 58、59、60、61、62、63、64、65、66、 67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、 82、 83、84、85、86、87、88、89、90、91、92、93、94、95、96、 97、98、99、100、101、102、103、104、105、 106、107、108、109、 110、111、112、113、114、115、116、117、118、119、120、121、 122、123、 124、125、126、127、128、129、130、131、132、133、 134、135、136、137、138、139、140、141、142、 143、144、145、 146、147、148、149、150、151、152、153、154、155、156、157、 158、159、160、 161、162、163、164、165、166、167、168、169、 170、171、172、173、174、175、176、177、178、179、 180、181、 182、183、184、185、186、187、188、189、190、191、192、193、 194、195、196、197、 198,199 or 200 amino acid, including all values and range, the section and amino acid sequence disclosed herein therebetween Any amino acid sequence have at least 80,85,90,95,96,97,98,99 or 100% identity.In some aspects, described One of the amino acid sequence for the HER3 binding antibody that amino acid section provides in such as table 6 or chart 1.
In other aspects, the antibody or polypeptide of embodiment of the present invention include appointing for amino acid sequence disclosed herein The amino acid section of what amino acid sequence, wherein the section start from amino acid position 1 in any sequence provided herein, 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25 to 25,26,27, 28、29、30、31、32、33、34、35、36、37、38、 39、40、41、42、43、44、45、46、47、48、49、50、51、52、 53、 54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、 69、70、71、72、73、74、75、76、77、 78、79、80、81、82、83、 84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、 99、100、101、 102、103、104、105、106、107、108、109、110、 111、112、113、114、115、116、117、118、119、120、 121、122、 123、124、125、126、127、128、129、130、131、132、133、134、 135、136、137、138、 139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、 158、 159、160、161、162、163、164、165、166、167、168、169、170、 171、172、173、174、175、 176、177、178、179、180、181、182、 183、184、185、186、187、188、189、190、191、192、193、194、 195,196,197,198,199 or 200 and the amino acid position 4 in the same sequence of the offer finally, 5,6,7,8,9, 10、11、12、13、14、15、16、17、 18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、 33、34、 35、36、37、38、39、40、41、42、43、44、45、46、47、 48、49、50、51、52、53、54、55、56、57、58、59、 60、61、62、 63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、 78、79、80、81、82、83、84、 85、86、87、88、89、90、91、92、 93、94、95、96、97、98、99、100、101、102、103、104、105、106、 107、108、109、110、111、112、113、114、115、116、117、118、 119、120、121、122、123、124、125、 126、127、128、129、130、 131、132、133、134、135、136、137、138、139、140、141、142、 143、 144、145、146、147、148、149、150、151、152、153、154、 155、156、157、158、159、160、161、162、 163、164、165、166、 167、168、169、170、171、172、173、174、175、176、177、178、 179、180、 181、182、183、184、185、186、187、188、189、190、 191、192、193、194、195、196、197、198、199 Or 200.In some aspects, the amino acid for the HER3 binding antibody that described section or part thereof provides in such as table 6 or chart 1 One of sequence.
In other aspects, the antibody or polypeptide of embodiment of the present invention include with V, VJ of HER3- binding antibody, VDJ, D, DJ, J or CDR structural domain (as provided in table 6 and chart 1) have at least 80,85,90,95,96,97,98,99 or 100% The amino acid section of identity (or may originate from any range therein).For example, polypeptide may include 1,2 or 3 with such as table 6 and The CDR1 of the HER3 binding antibody provided in chart 1,2 and/or 3 have at least 80,85,90,95,96,97,98,99 or 100% The amino acid section of identity (or may originate from any range therein).
It can apply for any other method described herein or composition in method and/or composition of the invention The embodiment discussed in context.Accordingly, with respect to the composition of a method embodiment be equally applicable to it is of the invention Other method and compositions.
As used in the description herein, " one (a) " or " a kind of (an) " can refer to one or more.As herein Used in the claims, when being used in combination with word "comprising", word " one (a) " or " a kind of (an) " can refer to one Or more than one.
Unless clearly indicate only alternative solution or alternative solution is mutual exclusion, otherwise term "or" in claim Using for meaning "and/or", although present disclosure is supported to refer to the definition of only alternative solution and "and/or".Such as institute herein With " another " can refer at least two or more.
In entire the application, term " about " for expression value include device, for measured value method constant error The variation for changing or being present between study subject.
It will be apparent according to the following detailed description other purposes of the invention, characteristic and advantageous aspect.However, Ying Li Solution, the detailed description and specific embodiment (although display the preferred embodiments of the invention) are only by way of illustration It provides, because of the variations and modifications skill common for this field according to this detailed description, in the spirit and scope of the present invention Art personnel will become obvious.
Brief description
Following drawings forms the part of this specification and is included to further illustration certain aspects of the invention.Pass through The detailed description of the specific embodiment in conjunction with shown in herein is better understood this with reference to the one or more of these attached drawings Invention.
The process flow of Fig. 1 antibody selection.
Fig. 2 is used for the Vector for Phage Display of rabbit Fab expression.
Anti- Her3 antibody on human that Fig. 3 is carried out using the Chinese hamster ovary celI for expressing heterologous HER3 and mouse HER3/ErbB3's In conjunction with.
Dose response of the anti-Her3IgG of Fig. 4 purifying in the inhibition measurement of HER3 phosphorylation.
The pAKT and pERK that Fig. 5 .HER3Mab IgG is generated in MCF7 cell inhibit.
Fig. 6 blocks the dose response of the anti-HER3 antibody of measurement display using the ligand of α screening format and chart.
Inhibition of the anti-HER3 antibody of Fig. 7 to the cell growth of the NRG induction of MCF7 cell.Data are shown as being led by antibody The percentage relative to no antibody control reduction caused.
The humanization HER3 monoclonal antibody that Fig. 8 is measured by ELISA combines the concentration dependent of HER3ECD.
The humanization HER3 monoclonal antibody that Fig. 9 is measured by Biacore combines the dynamics of HER3ECD affine Power.
Figure 10 humanization HER3 monoclonal antibody inhibits the concentration dependent of pHER3 in T47D cancer cell.
Figure 11 humanization HER3 monoclonal antibody inhibits the concentration dependent of CWR22 cancer cell multiplication.
Figure 12 humanization HER3 monoclonal antibody inhibits the concentration dependent of MCF-7 cancer cell multiplication.
The description of illustrative embodiment
I. antibody of the invention
In certain embodiments, it is contemplated that in conjunction with HER3 albumen at least partly and inhibit HER3 signal transduction and cancer The antibody of cell Proliferation or its segment.As used herein, term " antibody " is intended to broadly refer to any immunoconjugator, such as IgG, IgM, IgA, IgD, IgE and genetically modified IgG, and the antibody CDR structural domain comprising retaining antigen-binding activity Polypeptide.The antibody is selected from chimeric antibody, affine mature antibody, polyclonal antibody, monoclonal antibody, humanized antibody, people Antibody or antigen binding antibody fragment or natural or synthesis ligand.
Preferably, anti-HER3 antibody is monoclonal antibody or humanized antibody.Actually herein shown in research shows that The CDR of the identification of HER3- binding antibody can be placed in people's frame, and resulting humanized antibody keeps the height for HER3 Affinity (Fig. 8-9).Importantly, humanized antibody also efficiently inhibits HER3 signal transduction (Figure 10) and is able to suppress The cancer cell of HER3 positive cancer cell replicates (Figure 11-12).Therefore, HER3 binding antibody provided herein, particularly source of people Change antibody, is the promising candidate of new anticancer therapeutic agent.
Therefore, it by known method and as described in this article, can produce for HER3 albumen, its respective epitope One or more epitopes or any aforementioned epitope conjugate (no matter such antigen or epitope be from natural origin separation also It is the synthesis of derivatives or variant of native compound) it is specific polyclonal or monoclonal antibody, antibody fragment and combination Structural domain and CDR (engineered forms including any aforementioned substances).Another kind variation is that wherein a heavy chain targets HER3 simultaneously And another heavy chain targeting different carcinoma cellular targets such as HER2, EGFR, IGF1R, cMet or other cell surface targets is double special The building of property antibody.
The example of antibody fragment suitable for embodiment of the present invention includes, but are not limited to: (i) is by VL、VH、CLWith CH1The Fab segment of structural domain composition；(ii) by VHAnd CH1" Fd " segment of structural domain composition；(iii) by the V of single antibodyLWith C H" Fv " segment of structural domain composition；(iv) by VH" dAb " segment of structural domain composition；(v) CDR region separated； (vi)F (ab') 2 segment, the divalent fragments thereof of the Fab segment connected comprising two；(vii) scFv molecule (" scFv "), wherein VHKnot Structure domain and VLStructural domain forms the peptide linker of binding structural domain by allowing two structural domains to combine and connects；(viii) double special Property scFv dimer (referring to United States Patent (USP) No.5,091,513)；(ix) double-chain antibody is constructed more by Gene Fusion Valence or polyspecific segment (U.S. Patent Application Publication 20050214860).Fv, scFv or double-chain antibody molecule can pass through incorporation Connect VHAnd VLThe disulphide bridges of structural domain are stablized.It also can produce miniantibody (Hu etc. of the scFv comprising being connected to CH3 structural domain People, 1996).
Antibody sample combination peptidomimetic is also contained in embodiment.Liu et al. people (2003) describes that " antibody sample combines quasi- Peptide " (ABiP), for as simplified antibody and with certain of longer serum half-life and less lengthy and tedious synthetic method The peptide of a little advantageous aspects.
It can be inoculated with antigen such as HER3 extracellular domain protein (amino acid 1-643 of NCBI accession number M34309) dynamic Object, to generate the antibody for for HER3 albumen being specificity.Frequently by antigen binding or another molecule is conjugated in enhance Immune response.As used herein, conjugate be any peptides for being incorporated into the antigen of the immune response for causing animal, it is more The substance of peptide, protein or non-proteinaceous.It includes from a variety of single antibodies that the antibody that antigen inoculation generates is responded in animal Generate a variety of non-equal molecules (polyclonal antibody) that bone-marrow-derived lymphocyte generates.Polyclonal antibody is the population mixture of antibody type, Each of the antibody type can recognize the different epitopes in same antigen.It gives for generating polyclonal antibody in animal Correct condition, most of antibody in animal blood serum will identify animal by the collective schedule on its immune antigenic compound Position.The specificity only selects identification those of target antigen or epitope antibody to further enhance by affinity purification.
Monoclonal antibody is that wherein each antibody molecule identification same epitope (is derived from because of all antibody-producting cells Single bone-marrow-derived lymphocyte system) single antibody.For generate the method for monoclonal antibody (MAb) generally along be used to prepare it is more The identical route of those of clonal antibody route starts.In some embodiments, by rodent such as mouse and rat For generating monoclonal antibody.In some embodiments, the cell of rabbit, sheep or the frog is for generating monoclonal antibody.Rat Using be it is well known and can provide it is certain it is advantageous for the use of.Mouse (for example, BALB/c mouse) is routinely used and leads to The stabilization fusions of high percentage are often provided.
Hybridoma technology includes single bone-marrow-derived lymphocyte and immortalization marrow from the mouse being previously immunized with HER3 antigen The fusion of oncocyte (normally mouse myeloma).The technology provides the side in amplification single antibody cellulation endless number generation Method, to can produce an unbounded quantity of identical antibody of structure (monoclonal antibody) with same antigen or epitope specificity.
Plasma B cell is isolated from the freshly prepared rabbit peripheral blood mononuclear cells for the rabbit being immunized, and further Selection is used for HER3 combination cell.After enriched antibody generates B cell, separable total serum IgE simultaneously synthesizes cDNA.It is amplifiable to be self-possessed The DNA sequence dna of the antibody variable region of chain and light chain is built into phage display Fab expression vector, and is transformed into large intestine bar Bacterium (E.coli).HER3 specific binding Fab can be selected by taking turns enrichment elutriation more, and it is sequenced.It can will select HER3 in conjunction with hit be expressed as in rabbit overall length IgG and using mammalian expression vector system by it in human embryo kidney (HEK293) it is expressed as rabbit/people's chimeric versions thereof in cell (Invitrogen), and utilizes fast protein liquid chromatography (FPLC) Separation unit is purified using protein G resin.
In one embodiment, antibody is chimeric antibody, for example, comprising migrating to nonhuman sequence, human sequence or source of people Change the antibody of the antigen-binding subsequences from non-human donor of sequence (for example, frame and/or constant domain sequence).It has developed Method comes the light chain and heavy chain constant domain of the similar structures domain substitution monoclonal antibody in employment source, to keep external anti- The variable region of body is complete.Alternatively, generating " complete people " monoclonal antibody in the transgenic mice of human immunoglobulin gene.? Development approach carrys out the constant region for immunoglobulin sequence for having rodent such as mouse and human amino acid sequence by recombination to construct The variable domains of monoclonal antibody are transformed into more person form.In " humanization " monoclonal antibody, only high change CDR comes Derived from mouse monoclonal antibody, and frame and constant region from human amino acid sequence (referring to United States Patent (USP) No.5,091, 513 and 6,881,557).It is believed that being used in the amino acid sequence found in the corresponding position of human antibody is replaced by rodent Feature antibody in amino acid sequence will treat use process in reduce unfavorable immune response a possibility that.It can also will be miscellaneous Tumor or other other cells experience genetic mutations for generating antibody or other changes are handed over, this can change or can not change by miscellaneous The binding specificity for the antibody for handing over tumor to generate.
It is (including humanization, embedding for generating monoclonal antibody in various animal species and for generating different type Close and complete people) the method for monoclonal antibody be well known in the present art and be very predictable.For example, following beauty State's patents and patent applications provide the enabled description of such method: U.S. Patent application No.2004/0126828 and 2002/ 0172677；With United States Patent (USP) No 3,817,837,3,850,752,3,939,350,3,996,345,4,196,265,4, 275,149、4,277,437、4,366,241、4,469,797、4,472,509、4,606,855、 4,703,003、4,742, 159、4,767,720、4,816,567、4,867,973、4,938,948、 4,946,778、5,021,236、5,164,296、5, 196,066、5,223,409、5,403,484、 5,420,253、5,565,332、5,571,698、5,627,052、5,656, 434、5,770,376、 5,789,208、5,821,337、5,844,091、5,858,657、5,861,155、5,871,907、 5,969,108,6,054,297,6,165,464,6,365,157,6,406,867,6,709,659； 6,709,873；6,753, 407；6,814,965；6,849,259；6,861,572；6,875,434 and 6,891,024.It is cited herein and herein All patents, patent application publication case and other publications the application is incorporated herein by reference.
Antibody can be generated from any animal origin (including birds and mammal).Preferably, the antibody is sheep, mouse (such as mouse and rat), rabbit, goat, cavy, camel, horse or chicken source.In addition, new technology allow develop human antibody and Human antibody is screened from people's combinatorial antibody library.For example, phage antibody expression technology allows the case where animal immune is not present Lower production specific antibody, such as in United States Patent (USP) No.6, described in 946,546 (it is incorporated herein by reference).These Technology is in Marks (1992)；Stemmer(1994)；Gram et al. (1992)；Barbas et al. (1994)；With Schier et al. (1996) it is further described in.
Can be expected completely the antibody for HER3 will have the function of in and/or offset HER3 regardless of animal species, Dan Ke The ability in other sources of grand cell line or antibody.Certain animal species may be less preferably for generating therapeutic antibodies , because they can be easier to cause allergic reaction caused by the activation (by the part " Fc ") because of complement system.However, Complete antibody can be by enzymatic digestion at " Fc " (complement combination) segment, and the antibody fragment with binding structural domain or CDR.The portion Fc The removing divided reduces a possibility that antigen antibody fragments will cause undesirable immune response, and therefore, the antibody pair of no Fc It can be preferably in preventative or therapeutic treatment.As described above, can also construct antibody, with become chimeric antibody or part or Fully human antibodies, with reduce or eliminate because to animal application generated in other species or with the sequence from other species Unfavorable immune consequence caused by the antibody of column.
Alternative variations are on one or more sites usually in protein containing an amino acid to another amino acid Exchange, and can be designed to adjust polypeptide the (funeral with or without other functions or property of one or more properties It loses).Substitution can be conservative, that is, an amino acid is substituted by the amino acid with similar shape and charge.Conservative substitution is It is known in the art that and including for example following variation: alanine to serine, arginine to lysine, asparagine to paddy ammonia Amide or histidine, aspartic acid to glutamic acid, cysteine to serine, glutamine to asparagine, glutamic acid to day It is aspartic acid, glycine to proline, histidine to asparagine or glutamine, isoleucine to leucine or valine, bright Propylhomoserin is to valine or isoleucine, lysine to arginine, methionine to leucine or isoleucine, phenylalanine to junket Propylhomoserin, leucine or methionine, serine to threonine, threonine to serine, tryptophan to tyrosine, tyrosine to color Propylhomoserin or phenylalanine and valine are to isoleucine or leucine.Alternatively, replace can be it is non-conservative so that polypeptide Function or activity be affected.Non-conservative changes generally include to replace residue with chemically distinct residue, such as use pole Property or electrically charged amino acid substitution nonpolarity or uncharged amino acid, vice versa.
Protein can be recombination or external synthesis.Alternatively, non-recombinant or recombinant protein can be separated from bacterium.Also set Think the bacterium containing such variant can be applied in composition and method.It therefore, there is no need to protein isolate matter.
Imagine in the composition, every ml has total polypeptide, peptide and/or the protein of about 0.001mg to about 10mg.Therefore, It is about, at least about or at most about 0.001 that the concentration of protein, which can be, in composition, 0.010,0.050,0.1,0.2,0.3, 0.4、0.5、0.6、0.7、 0.8、0.9、1.0、1.5、2.0、2.5、3.0、3.5、4.0、4.5、5.0、5.5、6.0、 6.5、 7.0,7.5,8.0,8.5,9.0,9.5,10.0mg/ml or more (or may originate from any range therein).In this, about, At least about or at most about 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23, 24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、 39、40、41、42、43、44、45、46、47、48、 49、50、51、52、53、 54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、 69、70、71、72、73、 74、75、76、77、78、79、80、81、82、83、 84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、 99 or 100% it can be antibody in conjunction with HER3.
The immunomodulatory moiety of antibody or preferably antibody in other oroteins or can be expressed as and other albumen by chemically conjugated Fusion protein.For the purpose of this specification and appended claims, the protein of all such fusions is included in antibody Or in the definition of the immunomodulatory moiety of antibody.
Embodiment provides antibody or antibody sample molecule for HER3, is connected at least one reagent and is sewed with forming antibody Close the polypeptide and peptide of object or payload.In order to enhance the effect of antibody molecule is as diagnosticum or therapeutic agent, routinely connect Or covalent bond or compound at least one required molecule or part.Such molecule or part may be, but not limited to, at least one Kind effector molecule or report molecule.Effector molecule includes the molecule with required active such as cellular cytoxicity activity.It has been attached In the non-limiting examples of the effector molecule of antibody include toxin, therapeutic enzyme, antibiotic, radiolabeled nucleotide etc..Phase Instead, report molecule is defined as any part that measurement can be used to be detected.It has been conjugated the report molecule in antibody Non-limiting example includes enzyme, radioactive label, haptens, fluorescent marker, phosphorescent molecules, chemiluminescent molecule, chromophore, hair Optical molecule, light affinity molecule, coloured particle or ligand such as biotin.
It is known in the art several methods for antibody being attached to or being conjugated in its conjugate fraction.Some attachments Method includes the use of metallo-chelate, uses such as organic sequestering agent such as diethylene-triamine pentaacetic acid acid anhydride (DTPA)；It is sub- Ethyl triamine tetraacethyl；The p- toluenesulfonamide of N-；And/or attach to four chloro- 3-6- diphenylglycolurils -3 of antibody.Monoclonal Antibody can also in the presence of coupling agent such as diphenylglycoluril or periodate with enzyme reaction.Exist in these coupling agents In the case where or prepared and fluorescein-labeled conjugate by being reacted with isothiocyanates.
II. the treatment of disease
The some aspects of embodiment of the present invention can be used for preventing or treating relevant to HER3 signal transduction disease or Illness.The signal transduction of HER3 can be weakened using any drug appropriate to prevent cancer cell multiplication.Preferably, substance of this kind It can be anti-HER3 antibody.
" treatment " and " treatment " refers to the purpose for the treatment benefit for obtaining disease or healthy related conditions to subject Application applies therapeutic agent or is operated to subject or physical therapy.For example, treatment may include that application drug is effective The antibody of the inhibition HER3 signal transduction of amount.
" subject " and " patient " refers to people or inhuman, such as primate, mammal and spinal animals.Specific In embodiment, the subject is people.
Such as entire use herein, term " treatment benefit " or " effective in treatment " refer to improvement or raising by Any effect of welfare of the examination person in terms of the therapeutic treatment of the patient's condition.This includes, but are not limited to the S or S of disease Frequency or severity.For example, the treatment of cancer may include, such as the reduction of tumor size, the reduction of tumor invasiveness, cancer life The prevention of reduction or the transfer of long rate.The treatment of cancer also can refer to the extended survival of the subject with cancer.
A. pharmaceutical preparation
When carrying out the clinical application of the therapeutic composition containing inhibiting antibody, usually valuably prepares and be suitable for The drug or therapeutic composition of desired application.In certain embodiments, pharmaceutical composition may include for example, at least about 0.1% reactive compound.In other embodiments, reactive compound may include for example, about 2% to about 75% or about 25% To about 60% Unit Weight, and can be from being originated from any range therein.
Embodiment of the present invention is applied (as liquid solution or suspension) advantageously in the form of Injectable composition Therapeutic composition；The solid form for being suitble to be configured to solution or suspension in a liquid before injection can also be prepared.Also These emulsifiable preparations.
Phrase " pharmaceutically or pharmacologically acceptable " refers to not to be generated when (as appropriate) is applied to animal such as people Adverse effect, allergic reaction or other adverse reactions molecular entity and composition.It include antibody or volume according to present disclosure The preparation of the pharmaceutical composition of outer active constituent is known to those skilled in the art.In addition, for dynamic Object (for example, people) application, it should be understood that preparation should meet sterile, pyrogenicity as FDA biological standard office requires, Safety and purity rubric.
As used herein, " pharmaceutically acceptable carrier " include any and whole aqueous solvents (for example, water, alcohol/ Aqueous solution, saline solution, parenteral medium, sodium chloride, woods grignard glucose etc.), non-aqueous solvent is (for example, third The organic ester of glycol, polyethylene glycol, vegetable oil and injectable, such as ethyl oleate), decentralized medium, coating, surfactant, Antioxidant, preservative (for example, antibacterium or antifungal agent, antioxidant, chelating agent and inert gas), isotonic agent, absorption Delayed-action activator, salt, drug, drug stabilizing agent, gel, adhesive, excipient, disintegrating agent, lubricant, sweetener, flavoring agent, dye Material, liquid and nutritional supplement, such similar material and combinations thereof, this is known in ordinary skill people 's.The pH and exact concentration of the various components in pharmaceutical composition are adjusted according to well known parameter.
Term " unit dose " or " dosage ", which refer to, is physically separated unit suitable for subject, each unit Containing be computed generate above discuss (that is, approach appropriate and therapeutic scheme) relevant desired reaction is applied to it The therapeutic composition of predetermined amount.Amount (according to the number and unit dose for the treatment of) to be administered depends on desired effect.To The actual dose of the composition of embodiment of the present invention of patient or subject's application can be according to body and physiologic factor such as Weight, age, health and the gender of subject, the type of disease to be treated, the degree of disease penetration, controlling of being previously or is currently being Effect, stability and the toxin of intervention, the special hair disease of patient, administration method and specific therapeutic substance are treated to measure.For example, agent Amount also may include about 1 μ g/kg/ weight of every application to about 1000mg/kg/ weight (such range includes dosage therebetween) or It is more and may originate from any range therein.In the non-limiting example of range that may originate from number listed herein, About 5 μ g/kg/ weight to about 100 mg/kg/ weight, about 5 μ g/kg/ weight to the range of about 500mg/kg/ weight etc. can be applied. The active constituent that measurement is used in the composition and suitable dosage of individual subjects by the doctor for being responsible for application under any circumstance Concentration.
Reactive compound can be prepared with for parenteral administration, for example, be formulated for by intravenous, intramuscular, subcutaneous or The even injection of intraperitoneal routes.Normally, such composition can be prepared as to liquid solution or suspension；It can also prepare suitable For the solid form of solution or suspension to be prepared after adding liquid before injection；And also emulsifiable preparation.
The suitable medicament forms used of injecting include sterile aqueous solution or dispersion；Comprising sesame oil, peanut oil or contain The preparation of water propylene glycol；With the sterile powder for sterile injection liquid or the extemporaneous preparation of dispersion.In all cases, institute The form of stating must be sterile and must have the mobility for reaching the degree that it can easily be injected.It should also make It makes and be stable under storage requirement, and preservative treatment must be carried out and made with the pollution for resisting microorganism such as bacterium and fungi With.
The composition of protein properties can be configured to neutral or salt form.Pharmaceutically acceptable salt includes and inorganic acid (such as hydrochloric acid or phosphoric acid) or such organic acid such as example acetic acid, oxalic acid, tartaric acid, mandelic acid formation sour addition Salt (is formed) with the free amine group of protein.The salt formed with free carboxy can also be derived from inorganic base such as hydroxide Sodium, potassium hydroxide, aqua ammonia, calcium hydroxide or iron hydroxide, and it is derived from such organic base such as isopropylamine, front three Amine, histidine, procaine etc..
Pharmaceutical composition may include containing such as water, ethyl alcohol, polyalcohol (for example, glycerol, propylene glycol and liquid macrogol Deng), the solvent or decentralized medium of its mixture and vegetable oil appropriate.Mobility appropriate can be for example by using coating Such as lecithin is maintained by granularity needed for maintaining and by using surfactant in the case of a dispersion.Micro- life Object effect prevent using various antibacterial agents and antifungal agent for example p-hydroxybenzoic acid, anesin, phenol, sorbic acid, Thimerosal etc. is realized.In many cases it is preferred to which ground includes isotonic agent, such as carbohydrate or sodium chloride.Injectable composition Extended absorption can be by being realized using delayed absorber such as aluminum monostearate or gelatin in the composition.
B. combination therapy
In certain embodiments, the compositions and methods of the invention involve the inhibition combined with second or other therapy Active antibody or antibody fragment for HER3 of the HER3 in cancer cell multiplication.Such therapy can be used for treating and be situated between with HER3 The relevant any disease of the cell Proliferation led.For example, the disease can be cancer.
The method and composition (including combination therapy) improve treatment or protecting effect, and/or enhance another anticancer Or the curative effect of anti-hyperproliferative therapy.It can be increased with the kill for effectively realizing required effect such as cancer cell and/or cell The combined amount for the inhibition grown provides therapeutic and Preventive Method and composition.This method may include by the cell and antibody or Antibody fragment and the contact of the second therapy.Can by tissue, tumour or cell and it is one or more comprising one or more reagents (that is, Antibody or antibody fragment or anticancer agent) composition or pharmaceutical formulation thereof, or by that will organize, tumour and/or cell and two Kind or more different composition or preparation contact, one of composition 1) antibody or antibody fragment be provided, 2) anticancer agent Or 3) antibody or antibody fragment and anticancer agent.Similarly, it is contemplated that can by such combination therapy and chemotherapy, radiotherapy, Operative treatment or immunotherapy are used in combination.
Term " contact " and " exposure ", when being used for cell, herein for describing nationality with by therapeutic building Body and chemotherapeutant or radiotherapy dose are delivered to target cell or direct and placement location the process with target cell.It is thin in order to realize Born of the same parents kill, such as two kinds of reagents are delivered to cell with the combined amount for effectively killing cell or it being prevented to divide.
Can before anticancer therapy, period, later or with various combined administration inhibiting antibodies.Application can have from simultaneously Interval to several minutes to a couple of days to several weeks.Wherein antibody or antibody fragment and antitumor and anticancer agent are provided individually to patient In embodiment, it will typically ensure that and do not terminated between the time delivered each time for quite a long time, still so as to two kinds of compounds Can so the effect being advantageously combined be generated to patient.In such cases, it is contemplated that can be provided for patient each other about 12 to 24 In hour or 72 hours, more specifically, the antibody therapy and anti-cancer therapies in about 6-12 hours each other.In some cases, May expect significant extended treatment period, wherein between respective application experience a couple of days (2,3,4,5,6 or 7) to several weeks (1,2, 3,4,5,6,7 or 8).
In certain embodiments, therapeutic process will continue 1-90 days or more (such range include between every other day Number).One kind can be applied in the 1st to the 90th day any day (such range includes interval number of days) or any combination thereof by imagining Reagent, and it is another in the 1st to the 90th day any day (such range includes interval number of days) or any combination thereof application Kind reagent.In one day (in the period of 24 hours), it is one or many reagent can be applied to patient.In addition, after therapeutic process, if Want there is a period of time for not applying anticancer therapy.Depending on the patient's condition of patient, their prognosis, resistance, health etc., A month of sustainable 1-7 days of the period and/or 1-5 weeks and/or 1-12 or longer time, (such range included interval Number of days).It is expected that repeating treatment cycle when necessary.
Various combinations can be used.For following Examples, antibody therapy is " A " and anti-cancer therapies are " B ":
A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B
B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A
B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A
The general side for the application that any compound or therapy of the invention will comply with for such compound is applied to patient Case considers the toxicity (if any) of reagent.Therefore, in some embodiments, there are monitorings to be attributable to combination therapy Toxicity the step of.
I. chemotherapy
Can embodiment according to the invention use many chemotherapeutants.Term " chemotherapy " refers to be controlled using drug Treat cancer." chemotherapeutant " is used for the compound or composition for meaning to apply in the treatment of cancer.These reagents or drug According to their active patterns in the cell, (such as whether they influence the cell cycle and in what effect stepwise cell week Phase) classify.Alternatively, can be based on its directly crosslinking DNA, the intercalation of DNA or by influencing nucleic acid synthesis induced chromosome and having silk The capability representation reagent of abnormal division.
The example of chemotherapeutant includes alkylating agent such as phosphinothioylidynetrisaziridine and cyclophosphamide；Alkylsulfonate, such as busulfan, Improsulfan and piposulfan；Aziridine, such as benzo DOPA, carbaxilquinone, Meturedepa and uredopa；Aziridine and first Base melamine, including hemel, three ethylene melamines, triethylenephosphoramide, triethylene thiophosphoramide and trihydroxy methyl three Poly cyanamid；Annona lactone (especially manaca is pungent and its octanone of Bradley for bubble)；Camptothecine (including synthetic analogues support pool replaces Health)；Bryostatin；callystatin；CC-1065 (including its Adozelesin, Carzelesin are similar with Bizelesin synthesis Object)；Cryptophycin (especially macrolide 1 and macrolide 8)；Dolastatin；Mostly card meter Xing (including synthetic analogues KW- 2189 and CB1-TM1)；Eleutherobin (eleutherobin)；Water ghost any of several broadleaf plants alkali；sarcodictyin；Spongistatin (spongistatin)；Mustargen, such as Chlorambucil, Chlornaphazine, cholophosphamide, Estramustine, ifosfamide, mustargen, Nitrobine hydrochloride, melphalan, novembichin, phenesterine, pennisetum mustard (Prednimustine), bent phosphorus Amine (Trofosfamide) and uracil mastard；Nitroso ureas, such as Carmustine, chlorozotocin, Fotemustine, Luo Mosi Spit of fland, Nimustine and reynolds nitrogen mustard；Antibiotic, such as Enediyne Antibiotic are (for example, Calicheamicin, especially Jia Liche Mycin γ lI and Calicheamicin ω I1)；Up to endomycin, including reach endomycin A；Diphosphonate, such as clodronate；Ai Sibo Mycin；And neoearcinostain chromophore and related chromoprotein Enediyne Antibiotic chromophore, aclacinomycin (aclacinomysins), D actinomycin D, Anthramycin, azaserine, bleomycin, act-C, carabicin, ocean Erythromycin, cardinophyllin, chromomycin (chromomycinis), dactinomycin D, daunorubicin, Detorubicin, 6- diazonium -5- oxygen Generation-L- nor-leucine, adriamycin (including morpholino Doxorubicin, cyano morpholinyl-adriamycin, 2- pyrrolino-doxorubicin and Deoxidation adriamycin), Epi-ADM, esorubicin, idarubicin, marcellomycin, mitomycin such as mitomycin C, wheat examine Phenolic acid, nogalamycin, olivomycin, Peplomycin, porfiromycin, puromycin, triferricdoxorubicin, rodorubicin, chain are black Rhzomorph, streptozotocin, tubercidin, ubenimex, Zinostatin and Zinostatin；Anti- metabolite, such as methotrexate (MTX) and 5 FU 5 fluorouracil (5-FU)；Folacin, such as denopterin, pteropterin and Trimetrexate；Purine analogue, such as fluorine reach Draw shore, Ismipur, thiapurine and thioguanine；Pyrimidine analogue, such as ancitabine, azacitidine, 6- azoturia Glycosides, Carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine and floxuridine；Androgen, such as Kapp testis Ketone, Masterone, epithioandrostanol, Mepitiostane and Testolactone；Anti- adrenal gland, such as mitotane and Trilostane；Folic acid Replenishers, such as folinic acid；Aceglatone；Aldophosphamideglycoside；Amino-laevulic acid；Eniluracil；Amsacrine； bestrabucil；Bisantrene；Edatrexate；Defosfamide；Demecolcine；Diaziquone；According to Buddhist ornithine；Elliptinium Acetate；Ai Bo Mycin；Etoglucid；Gallium nitrate；Hydroxycarbamide；Lentinan；lonidainine；CHROMATOGRAPHIC FRACTIONATION AND MASS, such as maytansine and An Si bacterium Element；Mitoguazone；Mitoxantrone；Mopidamol (mopidanmol)；nitraerine；Spray department statin；Phenamet；The soft ratio of pyrrole Star；Losoxantrone；Podophyllic acid；2- ethylhydrazide；Procarbazine；PSK polysaccharide compound；Razoxane；Nitragin；Sizofiran； Spirogermanium；Tenuazonic acid；Triethyleneiminobenzoquinone；2,2 ', 2 "-ethylaluminum amine；Trichothecenes (especially T-2 poison Element, myconomycin A, Roridine A and anguidin)；Urethane；Long fields for spring sowing are new；Dacarbazine；Mannomustine；Dibromo is sweet Reveal alcohol；Mitolactol；Pipobroman；gacytosine；Arabinoside (" Ara-C ")；Cyclophosphamide；Taxanes, example Such as, taxol and Docetaxel gemcitabine；6- thioguanine；Mercaptopurine；Platinum coordination complex, such as cis-platinum, Ao Shali Platinum and carboplatin；Vincaleukoblastinum；Platinum；Etoposide (VP-16)；Ifosfamide；Mitoxantrone；Vincristine；Vinorelbine；Promise disappears Clever (novantrone)；Teniposide；Edatrexate；Daunorubicin；Aminopterin；Xeloda；Ibandronate；Irinotecan (for example, CPT-11)；Topoisomerase enzyme inhibitor RFS 2000；Difluoromethylornithine (DMFO)；Retinoid such as regards Yellow acid；Capecitabine；Carboplatin, procarbazine, plicamycin, gemcitabine, Noviburn, farnesyl protein transferase inhibitor, Inhibition of farnesyl protein transferase, the pharmaceutically acceptable salt of trans- platinum and any of above substance, acid or derivative.
Ii. radiotherapy
Cause DNA damage and the other factors that are widely used include commonly referred to as gamma-radiation, X-ray and/or Radioactive isotope to tumour cell direct delivering.Also contemplate the other forms of DNA impairment factor, such as microwave, proton Beam irradiates (United States Patent (USP) 5,760,395 and 4,870,287) and UV- irradiation.Most probably all of these factors taken together all to DNA, Precursor to DNA, the duplication to DNA and reparation and the assembly and maintenance of chromosome are caused widely to damage.X-ray Dosage range is the daily dose of 50 to 200 roentgens of (3 to 4 week) for a long time to the single dose of 2000 to 6000 roentgens. Radioisotopic dosage range variation is broad, and depends on the radiation intensity and type of the half-life period of isotope, transmitting And by the absorption of neoplastic cell.
Iii. immunotherapy
It will be appreciated by the skilled addressee that can combine other immunotherapy or combine with the method for embodiment It uses.In the background for the treatment of of cancer, immunotherapeutic agent, which is often relied on using immune effector cell and molecule, to be targeted and breaks Bad cancer cell.RituximabIt is such a example.Immune effector can be for example thin for tumour Some markers on cellular surface are the antibody of specificity.The antibody individually effector used as a treatment or its can raise Other cells actually realize cell killing.Antibody can be also conjugated in drug or toxin (chemotherapeutant, radioactive nucleus Element, ricin A chain, cholera toxin, pertussis toxin etc.) and it is used only as targeting agent.Alternatively, the effector can be and take The lymphocyte for the surface molecular that band is directly or indirectly interacted with tumour cell target.Various effector cells include cytotoxicity T cell and NK cell.
In the one aspect of immunotherapy, tumour cell must have it is some be suitable for targeting, that is, be not present in most of Marker on other cells.Many tumor markers exist and any of these markers is suitably adapted for of the invention It is targeted in the background of embodiment.Common tumor markers include CD20, carcinomebryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis antigen, MucA, MucB, PLAP, laminin receptor, erb B and p155.Exempt from The another aspect of epidemic disease therapy is combination antitumaous effect and immunostimulation.There is also molecules of immunization stimulus, including cell factor, Such as IL-2, IL-4, IL-12, GM-CSF, γ-IFN, chemotactic factor (CF), such as MIP-1, MCP-1, IL-8 and growth factor, it is all Such as FLT3 ligand.
The example for being currently in research or the immunotherapy in is immunologic adjuvant, for example, mycobacterium bovis BCG (Mycobacterium bovis), plasmodium falciparum (Plasmodium falciparum), dinitrofluorobenzene and aromatic series Close object (United States Patent (USP) 5,801,005 and 5,739,169；Hui and Hashimoto, 1998；Christodoulides et al., 1998)；Cytokine therapy, for example, interferon-' alpha ', β and γ, IL-1, GM-CSF and TNF (Bukowski et al., 1998； Davidson et al., 1998；Hellstrand et al., 1998)；Gene therapy, for example, TNF, IL-1, IL-2 and p53 (Qin etc. People, 1998；Austin-Ward and Villaseca, 1998；United States Patent (USP) 5,830,880 and 5,846,945)；It is anti-with monoclonal Body, for example, anti-CD 20, anti-Ganglioside GM2 and anti-p185 (Hollander, 2012；Hanibuchi et al., 1998； United States Patent (USP) 5,824,311).One or more anti-cancer therapies can be used together by imagination with antibody therapy described herein.
Iv. it performs the operation
About 60% people with cancer will undergo the operation of same type, the operation include it is preventative, diagnostic or Property, the operation of curative and palliative by stages.Curative operation includes that all or part of of wherein cancerous tissue is removed by physics, cuts The excision for removing and/or destroying, and can be treated with the treatment, chemotherapy, radiation of other therapies such as embodiment of the present invention Method, hormonotherapy, gene therapy, immunotherapy and/or alternative medicine are used in combination.Tumor resection refers to tumour at least partly Physical removal.Other than tumor resection, the treatment using operation includes laser surgey, cryosurgery, electrosurgery and use The operation (mohs' technique) of microscope control.
After the excision of some or all of cancerous cells, tissue or tumour, chamber can be formed in vivo.The treatment can pass through The region is irrigated with other anti-cancer therapies, direct injection or the local topical application of drug are realized.Can for example every 1,2,3, 4、5、6Or 7 days or every 1,2,3,4 and 5 week or every 1,2,3,4,5,6、7、8, 9,10,11 or 12 months repetition such treatments.This A little treatments can equally have different dosage.
V. other reagents
Imagining some aspects of other reagents and embodiment of the present invention can be applied in combination to improve the treatment for the treatment of Effect.These other reagents include realizing that cell surface receptor is connected with GAP, cell growth inhibition and differentiation agent, cell glue Inhibitor, enhancing excessive proliferated cell it is upper to the reagent of the sensibility of cell death inducer or other biological reagents It adjusts.The increase for the cell signalling that number by increasing GAP connection generates can enhance the cell mass to adjacent hyper-proliferative The anti-hyper-proliferative of body acts on.In other embodiments, cell can be inhibited or differentiation agent and embodiment of the present invention The effect of to improve the anti-hyper-proliferative for the treatment of is applied in combination in some aspects.The inhibitor of imagination cell adhesion improves of the invention The effect of embodiment.The example of cell adhesion inhibitors is focal adhesion kinase (FAK) inhibitor and Lovastatin.It is contemplated that can Excessive proliferated cell will be enhanced to other reagents such as antibody c225 and embodiment of the present invention of the sensibility of natural death of cerebral cells Some aspects combine to improve therapeutic efficiency.
III. kit and diagnosticum
In the various aspects of embodiment, it is contemplated that reagent contains therapeutic agent and/or other therapeutic agents and delivery of agents.One In a little embodiments, embodiment of the present invention is related to being used to prepare and/or applying the kit of the therapy of embodiment.Institute State the sealed vial that kit may include one or more any pharmaceutical compositions equipped with embodiment of the present invention.The examination Agent box may include for example, at least a kind of HER3 antibody and preparation, preparation and/or apply the component of embodiment or carry out this hair The reagent of the one or more steps of bright method.In some embodiments, the kit also may include container appropriate, It is will not be with the container of the component reaction of kit, such as eppendorf pipe, assay plate, syringe, bottle or pipe.It is described Container can be by can sterilising material such as plastics or glass manufacture.
The kit may also include summarize herein shown in method process step specification, and would be complying to The substantially the same method of method described herein is known to those skilled in the art.The explanation Letter breath may be present in the computer-readable medium containing machine readable instructions, described instruction, when being executed using computer, The true or practical process of the therapeutic agent of display delivering medicine effective quantity.
IV. embodiment
The following example is included to display the preferred embodiments of the invention.Those of ordinary skill in the art should manage It solves, technology disclosed in embodiment follows the operational excellence in the practice of the invention being discovered by the present inventors, so as to be recognized To constitute the representative art for its preference pattern implemented.However, those of ordinary skill in the art are according to present disclosure It should be understood that many variations can be generated in the specific embodiments which are disclosed without departing from the spirit and scope of the present invention And still obtain similar or like result.
Embodiment 1- uses the HER3 antigen binding plasma B cell for the rabbit separation being immunized from antigen from phage display Fab Library generates rabbit monoclonal antibodies
ErbB3 (Genebank accession number M34309) extracellular domain (ECD) (amino acid Met1-Thr643) albumen For new zealand rabbit to be immunized in Bethyl laboratories, Inc (Montgomery, TX).It is immune with the administration of 100 μ g/ rabbits Rabbit.After initial immunity, 2 reinforcements administration was applied with 2-3 weeks dosing interval.By the way that HER3ECD albumen is coated in 96 holes On plate (max-sorb plate, Nunc), anti-Her3 serum is measured using a series of serum dilutions in ELISA, and utilize It is conjugated with the anti-rabbit antibody and tmb substrate detection binding antibody of horseradish peroxidase (HRP).By the absorbance at 450nm For measuring antibody serum titer.When titre reaches > 106When, whole blood sample is taken out from immune rabbit, and thin using fluoroscopic assist Born of the same parents sort (FACS) instrument (BD FACSAriaTMIII, BD Biosciences) divide from freshly prepared rabbit peripheral blood mononuclear cells From plasma B cell (CD45+CD5-CD19+).The further HER3 combination cell in the isolated plasma cell of selection.It is raw in antibody After being enriched at B cell, total serum IgE is separated, and according to manufacturer's recommendation, uses superscript reverse transcriptase II (Invitrogen) cDNA is synthesized.It is expanded using the primer (table 1) of one group of design by polymerase chain reaction (PCR) to be self-possessed The DNA sequence dna of the antibody variable region of chain and light chain, see, e.g., Ridder et al., 2001 (being incorporated herein by reference).It will The DNA of amplification is built into phage display Fab expression vector (Fig. 2) and is transformed into Escherichia coli (TG1) cell.In phage display technology Show and constructs 10 in carrier system8-9Library size, elutriation be enriched with by two-wheeled select HER3 and specifically bind Fab, and to it (Lone Star Labs, TX) is sequenced.The method flow for antibody selection has been illustrated in Fig. 1.
Selected HER3 can be expressed as in rabbit overall length IgG in conjunction with hit, and use mammalian expression vector system It is expressed as to rabbit/people's chimeric versions thereof in human embryo kidney (HEK293) cell (Invitrogen), and utilizes fast protein liquid Phase chromatography (FPLC) separative unit, is purified using protein G resin.Characterize the various biology of the HER3 antibody of purifying Matter.
Table 1. is used for the PCR primer group from rabbit plasma B cell clone variable heavy chain and light chain cDNA sequences.
Embodiment 2-measures the combination of people and mouse HER3/ErbB3 using flow cytometry and ELISA
By CHO Flp-in (Invitrogen) cell line for expressing people or mouse HER3 receptor for studying HER3 antibody Combination to cell surface receptor.Using without enzyme EDTA dissociation solution separation expression HER3 cell and will be with about 1x106 The concentration of a cell/ml is resuspended to PBS, in 2%FBS.Dyeing 40 is carried out to cell with the HER3IgG of purifying at 4 DEG C Minute, by being washed cell 2 times in the PBS containing 2%FBS within 10 minutes with 1200rpm centrifugation.Remove supernatant, 4 DEG C with The anti-rabbit igg of R-PE is conjugated with to cell dyeing 30 minutes.Cell then is washed with the PBS that 4mL contains 2%FBS, in streaming The cell is analyzed on cell instrument (Quava, Millipore).Fig. 3 shows that rabbit HER3 antibody on human and mouse HER3 expression are thin The combination of born of the same parents, as indicated by the increase by fluorescence intensity (X- axis) compared with being compareed in conjunction with non-specific rabbit igg.
Embodiment 3-is combined using Biacore T-100 using the HER3 that surface plasma body resonant vibration (SPR) measurement carries out The measurement of affinity
All experiments are carried out at 25 DEG C with 45 l/ minutes flow velocitys of μ.In order to prepare BIAcore measurement, using such as by manufacturing Anti- rabbit igg antibody (respective 50 μ g/ml, in acetate buffer, pH 5.0) is fixed on carboxylic first by the amine coupling method of quotient's description On base glucan sensor chip (CM5).By the rabbit Mab of purifying to be tested with the concentration dilution of 5 μ g/ml in 0.5%P20, In HBS-EP buffer, and it is injected on FC2 to reach 500 to 1000RU.FC1 is used as reference cell.Specific signal pair The difference of the opposite signal obtained on FC1 of the signal that Ying Yu is obtained on FC2.In 0.5%P20, HBS-EP buffer Series of concentrations dilution (100,50,25,12.5,6.25 and 3.13,1.56nM) within 90 second time injection of analytes (recombination People HER3, the apparent molecular weight on PAGE gel are 97 kDa).This is prepared from stoste in 0.5%P20, HBS-EP A little concentration.In the dissociation phase of 30 minutes monitoring in time analytes.Also to inject electrophoresis under the same conditions with double references slow Fliud flushing.After each cycle of operation, the Glycine-HCl buffer pH 1.5 by injecting 20 to 45 μ l regenerates two kinds of flowings Cell.To the combination K of HER3DIt is calculated by the koff/kon kinetic rate of each HER3 monoclonal antibody (table 2).
The HER3 antibody knot to people's HER3/ErbB3 extracellular domain that table 2. is measured using BIAcore (SPR) analysis Close kinetic constant
The monoclonal HER3 antibody of purifying KD(nM)
Rab46 73±0.13
Rab1210 1.0±0.05
Rab189 3.6±0.16
Rab774 0.16±0.01
The internalization research of embodiment 4-HER3 monoclonal antibody
By converging for T47D breast cancer cancer cell culture to 80%.Cell is separated, is resuspended in complete medium To count cell.In the case where the HER3IgG of 2 μ g/ml exists and is not present in 37 DEG C of incubation concentration about 1x107A/ml's Cell carries out 3 hours.Cell is washed with 4mL PBS, 2%FBS, and is combined using HER3 antibody (10 μ g/ml) as level-one Antibody dyes 40 minutes at 4 DEG C.Goat anti-rabbit antibodies (the BD of R-PE is being conjugated with 0.5 μ g after 2%FBS washing with PBS Pharmingen) at 4 DEG C to cell dyeing 30 minutes, the cell is covered with foil.Cell is washed with 4mL PBS, 2%FBS, Then with 1200rpm centrifugation 10 minutes.Cell is fixed in PBS/10% formaldehyde, and uses flow cytometer (Quava, Millipore) is analyzed.The HER3 by isolated HER3 mediated monoclonal antibody is calculate by the following formula by internal The percentage of change: the MFI X of (MFI of the average fluorescent strength (MFI) of no antibody control-antibody processing cell)/control 100, it is shown in table 3.
Table 3. by anti-HER3 antibody-mediated HER3 receptor internalisation percentage
HER3 monoclonal antibody The percentage of receptor internalisation
Rab46 39±2
Rab1210 62±5
Rab189 55±4
Rab774 60±8
Inhibition of the embodiment 5-rabbit HER3 monoclonal antibody to pHER3, pAKT and pERK1/2
By MCF7 cell with about 3x105The cell density of a cell/ml is seeded in 96 orifice plates, and overnight incubation.It will be thin Born of the same parents' culture medium is replaced with low serum (0.5%FBS), carries out 15 hours, is handled at 37 DEG C using isolated HER3 monoclonal antibody Cell 2 hours.With 3.3nM rhNRG1- β 1 37 DEG C stimulation cell 20 minutes, then generate cell pyrolysis liquid.Generating cell Before lysate, washed cell 3 times with cold PBS, 0.5%BSA.Cell is added gently shaking lower into each hole at 4 DEG C Extraction buffer (contains fresh 1mM PMSF, protease inhibitor cocktail and phosphatase inhibitor cocktail), carries out 30 Minute.After being mixed lysate by upper and lower liquid relief 3-5 times, plate is centrifuged 10 minutes with 3,000rpm, in following measurement Use cell supernatant.
PHER3 is inhibited to measure, is coated in 4 DEG C of mouse Anti-Human HER3 antibody (R&D Systems) with 4 μ g/ml Maxi-sorp plate (96 orifice plate of Costar) is stayed overnight, at room temperature with PBS closed plate 2 hours containing 2%BSA.By 100 μ l's Cell supernatant is transferred to closed assay plate, and is incubated for 2 hours in RT.Contain the PBS of Tween 20 (0.05%) with 300 μ l Washing plate 5 times.Then the addition anti-p- tyrosine-HRP of secondary antibody (R&D Systems) and it is incubated for 1 hour at RT, then It is detected.Step is washed repeatedly, is gently shaking lower addition HRP chemiluminescent substrate (Millipore, CA) at RT, into Row 5 minutes, luminous signal then was read using plate reader (Molecular Devices, CA).PHER3 is calculated using following formula Inhibition percentage: (signal of the cell of no antibody processing-signal of the cell of antibody processing)/the signal X 100 of control. 3-4 duplicate average values inhibit measurement (Fig. 4) for concentration dependent.Using quasi- using GraphPad prism (v5.1) The IC of the titration curve measurement of conjunction50It is shown in Table 4.
PAKT is inhibited to measure, using the antibody from R&D system, similar ELISA format is used to capture always AKT albumen.After washing plate 5 times with the PBS-T in 300 holes μ l/, secondary antibody biotinylation rabbit Anti-Human's phosphorus-Akt is added (S473) to be incubated for 1 hour at RT.By being incubated for 20 minutes at RT, streptavidin-HRP is used to detect. After washing plate, HRP chemiluminescent substrate is added, is carried out 5-10 minutes, and uses plate reader (Molecular Devices) Read plate.The percentage of the inhibition of pAKT is calculated using following formula: (the signal of the cell of no antibody processing-antibody processing cell Signal)/control signal X 100.3-4 duplicate average values inhibit measurement (Fig. 5) for concentration dependent.Use utilization The IC of the titration curve measurement of GraphPad prism (v5.1) fitting50It is shown in Table 4.
PERK1/2 is inhibited to measure, closes Meso Scale with the TRIS containing 3%BSA in the case where shake The plate of Discovery (MSD) precoating.Cell pyrolysis liquid (25 hole μ l/) is transferred to closed assay plate, and in shake In the case of be incubated for 2 hours at RT.With Tris buffer washing hole 3 times of 300 holes μ l/, and according to manufacturer's recommendation, benefit With the anti-phosphorus-ERK1/2 (T/Y:202/204 of MSD from MSD assay kit；185/187) antibody and SULFO-TAGTMIt is molten Liquid is detected.In MSD SECTORTMPlate is read on Imager 2400, and is analyzed using following formula: (no antibody processing The signal of cell-signal of the cell of antibody processing)/the signal X 100 compareed.3-4 duplicate average values for concentration according to Property is relied to inhibit measurement (Fig. 5).Use the IC of the titration curve measurement using GraphPad prism (v5.1) fitting50It is shown in table 4 In.
Combination of the embodiment 6-HER3 monoclonal antibodies block ligand neuregulin to HER3
HER3 ligand neuregulin (NRG1) may be incorporated on HER3 extracellular domain and activate HER3 phosphoric acid Change and downstream signal transduction.Selected rabbit HER3 monoclonal antibodies block ligand is measured to HER3 using α screening format measurement Combination.Biotinylation is carried out to NRG1 (R&D Systems) using biotinylation kit (Fisher Scientific), And HER3ECD is marked with 10 histidines (His).In opaque/white half hole face plate (Costar), by purifying Anti- HER3Mab is serially diluted to by 25mM Hepes, 100 mM NaCl, 0.5%BSA and 0.05%Tween20 with 2 times and is prepared Measurement buffer in.Rabbit igg 1 is used as negative control.With the concentration of 10nM into the assay plate containing the antibody being serially diluted Continuous addition biotinylation NRG-1 beta ligands and Her3/His10Receptor.It is being incubated at room temperature receptor in the case where gently shaking, matching Mixture 90 minutes of body and antibody.By the anti-strepto- biotin protein donor bead of AlphaScreen and His- nickel acceptor bead It is added in measurement with the final concentration of respective 20 μ g/ml.With foil overlay measurement plate, it is being incubated at room temperature in the case where gently shaking 1 hour.Plate is read on EnVision plate reader.HER3 monoclonal antibodies block NRG1 ligand shows the combination of HER3 Concentration dependent (Fig. 6) is fitted using four parameter curves and concentration titrations curve graph using GraphPad to estimate IC50It is worth (table 4)。
The anti-HER3 antibody that table 4. is assessed from dose dependent titration curve to the inhibition of the inhibition of pHER3, pAKT, The IC50 of the inhibition of pERK and the inhibition to NRG ligand binding
Inhibition of the anti-HER3mAb of embodiment 7- to cancer cell multiplication
In order to measure inhibition of the HER3 antibody of separation to cancer cell multiplication, by MCF7 (the human milk gland of 3000 cells/wells Cancer) 100 μ ls of the cancer cell inoculation on 96 orifice plates 10%FBS culture medium in, in 37 DEG C of incubators overnight.With containing The 0.5%FBS culture medium substitutive medium of HER3 antibody, and stimulate with 100ng/ml β-NRG (by directly adding ligand To in culture medium antibody-containing) in the case where cultivate 72 hours.By by the AlamarBlum of 10 μ lTM(Invitrogen) It is added in culture medium to measure cell Proliferation, cell is incubated in 37 DEG C of incubators, in addition AlamarBlumTM (excite in 535nm and emit in 590nm) 2 hours measurement fluorescence signals afterwards.By the NRG grown in MCF7 cell induction The inhibition of cell growth is calculated as the percentage (Fig. 7) for the reduction that antibody processing is handled relative to no antibody, and IC50Value is listed in In table 5.
IC50 of the anti-HER3 monoclonal antibody of table 5. to the inhibition of cancer cell multiplication.
HER3 monoclonal antibody IC50 (95% confidence level), nM
Rab46 15.28 to 42.28
Rab1210 11.92 to 28.53
Rab189 31.66 to 86.07
Rab774 151.2 to 526.7
The DNA and amino acid sequence of antibody variable region is sequenced in embodiment 8-
IgG light chain variable region (LV) and heavy chain variable region (HV) are sequenced using Genewiz (Edison, NJ).Gently Chain is variable and heavy chain variable amino acid sequence is all listed in Table 6 below.It is described using the CDR of IMGT program analysis light chain and heavy chain CDR is listed in Table 6 below.
The anti-HER3 antibody sequence of table 6.
The humanization of embodiment 9- rabbit HER3 monoclonal antibody
It is identified using IMGT database (Lafranc et al., 2012, be incorporated herein by reference) and is most matched with rabbit VH and VL People's VH and VL Germline sequences.Humanization is based on CDR transplanting concept (Haidar et al., 2012), utilizes Kabat/Chothia's Combine (Yu et al., 2010 and Haidar et al. 2012 (referring also to Retter et al., 2005 and Singer et al., 1993)) boundary Determine the CDR of rabbit antibody.
2 variable heavy chain sequences and 2 light chain variable sequences in total are designed for each anti-HER3 monoclonal antibody: Such as the following Rab46 shown in chart 1 and Rab1210 (CDR sequence is subject to underscore).
Chart 1: the variable domains of humanized antibody.
The building and generation of Humanized monoclonal antibodies
The DNA sequence dna of the humanization variable region from design is synthesized, and is cloned into as described in preceding embodiment IgG1 expression vector, for generating humanized antibody in HEK293 cell.By recombinating 1 heavy chain in each antibody 8 Humanized monoclonal antibodies in total are produced with 1 light chain.The heavy chain of each humanized antibody and the composite column of light chain In following table 8.
Table 8: the heavy chain and light chain variable region of humanization HER3mAb
The title of humanized antibody Parental clones title Variable heavy chain sequence Light chain variable sequence
HER3-hMab-A9 Rab46 hRab46H-1 hRab46L-1
HER3-hMab-A10 Rab46 hRab46H-2 hRab46L-1
HER3-hMab-A11 Rab46 hRab46H-1 hRab46L-2
HER3-hMab-A12 Rab46 hRab46H-2 hRab46L-2
HER3-hMab-A13 Rab1210 hRab1210H-1 hRab1210L-1
HER3-hMab-A14 Rab1210 hRab1210H-2 hRab1210L-1
HER3-hMab-A15 Rab1210 hRab1210H-1 hRab1210L-2
HER3-hMab-A16 Rab1210 hRab1210H-2 hRab1210L-2
The characterization of the anti-HERS monoclonal antibody of humanization
Combination of the humanization HER3 monoclonal antibody to the extracellular domain (ECD) of HER3 is measured by ELISA:It is all Monoclonal antibody shows the strong combination to people HER3ECD similar with parental generation rabbit monoclonal antibodies, as shown in Fig. 8 A.For The group of 4 humanized antibodies from parental generation Rab46, all 4 antibody retain the combination to mouse HER3ECD structural domain Affinity (Fig. 8 B).The EC of the estimation of those humanized antibodies50(concentration generates 50% maximum binding capacity) is listed in Table 9 below.
Table 9: pass through the EC of the humanization HER3 antibody of ELISA measurement50(μg/ml)。
Pass through the HER3 antigen binding dynamics for the humanization HER3 antibody that surface plasma body resonant vibration (SPR) method measures: Use the kinetic constant of T-100Biacore instrument (table 10) measurement antibody.Humanization HER3 antibody and its parental generation rabbit/people Fc are embedding Fit SPR sensorgram is shown in Figure 9.
Table 10: the kinetic association constant of humanization HER3 antibody.
Antibody ka(1/Ms) kd(1/s) KD(M)
A10 1.41E+05 1.69E-04 1.20E-09
A14 2.01E+05 1.74E-04 8.62E-10
CHI46 1.38E+05 4.58E-04 3.32E-09
CHI1210 5.38E+05 6.44E-04 1.20E-09
Inhibition of the humanization HER3 monoclonal antibody to HER3 phosphorylation:As in preceding section for rabbit HER3 antibody institute Description, carry out the measurement of the inhibition of HER3 signal transduction.As shown in Figure 10, humanization HER3 antibody A 10 and A14 are shown It is equal with their parental generation antibody, than their better inhibition to HER3 phosphorylation.From concentration titrations curve graph (figure 10) IC that the pHER3 of estimation inhibits50It is shown in Table 11.
Table 11: IC of the humanization HER3 antibody to the estimation of the inhibition of pHER350。
Inhibition of the humanization HER3 monoclonal antibody to cancer cell multiplication: use with for utilize rabbitMonoclonal antibody is ground Study carefully the inhibition that the identical method of described method carries out cancer cell multiplication.Using two cancerous cell lines, one is breast cancer cell It is MCF-7, another cell line is prostate cancer cell line CWR22, and two cell lines come from ATCC (American tissue culture Center).The positive correlation of the inhibition display and increased antibody concentration of cancer cell multiplication, as shown in Figure 11 and 12.Estimation The inhibition to cancer cell multiplication IC50It is shown in Table 12.
Table 5: IC of the humanization HER3 antibody to the estimation of the inhibition of cancer cell multiplication50。
* ND, it is undeterminate
***
According to present disclosure, disclosed herein and statement all methods are can produce and executed without excessive reality It tests.Although the compositions and methods of the invention are described by preferred embodiment, for ordinary skill people Member it is apparent that can change without departing substantially from the concept, spirit and scope of the present invention method described herein and The sequence of the step of method or step.More specifically, it is apparent that available in chemical and physiologically relevant certain reagents In substitution reagent described herein, while it can get the same or similar result.To those skilled in the art Be obvious all such similar substitutions and modification be considered the spirit of the invention being such as defined by the appended claims, In range and concept.
Reference
Following reference provides illustrative methods at them or is supplemented in detail the other of method shown in herein In degree, explicitly by being incorporated herein by reference.
U.S. Patent application No.2002/0172677；2004/0126828 and 20050214860
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Claims (15)

1. a kind of isolated monoclonal antibody, the antibody specificity combination HER3, also, in the antibody:
(I)
(a) the first VHCDR is identical as SEQ ID NO:15；
(b) the 2nd VHCDR is identical as SEQ ID NO:16；
(c) the 3rd VHCDR is identical as SEQ ID NO:17；
(d) the first VLCDR is identical as SEQ ID NO:18；
(e) the 2nd VLCDR is identical as SEQ ID NO:19；And
(f) the 3rd VLCDR is identical as SEQ ID NO:20；
(II)
(a) the first VHCDR is identical as SEQ ID NO:9；
(b) the 2nd VHCDR is identical as SEQ ID NO:10；
(c) the 3rd VHCDR is identical as SEQ ID NO:11；
(d) the first VLCDR is identical as SEQ ID NO:12；
(e) the 2nd VLCDR is identical as SEQ ID NO:13；And
(f) the 3rd VLCDR is identical as SEQ ID NO:14；
(III)
(a) the first VHCDR is identical as SEQ ID NO:21；
(b) the 2nd VHCDR is identical as SEQ ID NO:22；
(c) the 3rd VHCDR is identical as SEQ ID NO:23；
(d) the first VLCDR is identical as SEQ ID NO:24；
(e) the 2nd VLCDR is identical as SEQ ID NO:25；And
(f) the 3rd VLCDR is identical as SEQ ID NO:26；Or
(IV)
(a) the first VHCDR is identical as SEQ ID NO:27；
(b) the 2nd VHCDR is identical as SEQ ID NO:28；
(c) the 3rd VHCDR is identical as SEQ ID NO:29；
(d) the first VLCDR is identical as SEQ ID NO:30；
(e) the 2nd VLCDR is identical as SEQ ID NO:31；And
(f) the 3rd VLCDR is identical as SEQ ID NO:32.
2. antibody according to claim 1, in which:
(a) the first VHCDR is identical as SEQ ID NO:15；
(b) the 2nd VHCDR is identical as SEQ ID NO:16；
(c) the 3rd VHCDR is identical as SEQ ID NO:17；
(d) the first VLCDR is identical as SEQ ID NO:18；
(e) the 2nd VLCDR is identical as SEQ ID NO:19；And
(f) the 3rd VLCDR is identical as SEQ ID NO:20.
3. antibody according to claim 1, in which:
(a) the first VHCDR is identical as SEQ ID NO:9；
(b) the 2nd VHCDR is identical as SEQ ID NO:10；
(c) the 3rd VHCDR is identical as SEQ ID NO:11；
(d) the first VLCDR is identical as SEQ ID NO:12；
(e) the 2nd VLCDR is identical as SEQ ID NO:13；And
(f) the 3rd VLCDR is identical as SEQ ID NO:14.
4. antibody according to claim 1, in which:
(a) the first VHCDR is identical as SEQ ID NO:21；
(b) the 2nd VHCDR is identical as SEQ ID NO:22；
(c) the 3rd VHCDR is identical as SEQ ID NO:23；
(d) the first VLCDR is identical as SEQ ID NO:24；
(e) the 2nd VLCDR is identical as SEQ ID NO:25；And
(f) the 3rd VLCDR is identical as SEQ ID NO:26.
5. antibody according to claim 1, in which:
(a) the first VHCDR is identical as SEQ ID NO:27；
(b) the 2nd VHCDR is identical as SEQ ID NO:28；
(c) the 3rd VHCDR is identical as SEQ ID NO:29；
(d) the first VLCDR is identical as SEQ ID NO:30；
(e) the 2nd VLCDR is identical as SEQ ID NO:31；And
(f) the 3rd VLCDR is identical as SEQ ID NO:32.
6. antibody according to claim 1, wherein the antibody includes:
(i) V as shown in SEQ ID NO:1, SEQ ID NO:33 or SEQ ID NO:34HStructural domain, and such as SEQ ID NO:2, V shown in SEQ ID NO:35 or SEQ ID NO:36LStructural domain；
(ii) V as shown in SEQ ID NO:3, SEQ ID NO:37 or SEQ ID NO:38HStructural domain, and such as SEQ ID NO: 4, V shown in SEQ ID NO:39 or SEQ ID NO:40LStructural domain；
(iii) V as shown in SEQ ID NO:5HStructural domain and the V as shown in SEQ ID NO:6LStructural domain；Or
(iv) V as shown in SEQ ID NO:7HStructural domain and the V as shown in SEQ ID NO:8LStructural domain.
7. antibody according to claim 6, wherein the antibody includes:
(i) V as shown in SEQ ID NO:33HStructural domain and the V as shown in SEQ ID NO:35 or SEQ ID NO:36LStructure Domain；
(ii) V as shown in SEQ ID NO:34HStructural domain and the V as shown in SEQ ID NO:36 or SEQ ID NO:35LStructure Domain；
(iii) V as shown in SEQ ID NO:37HStructural domain and the V as shown in SEQ ID NO:39 or SEQ ID NO:40LKnot Structure domain；Or
(iv) V as shown in SEQ ID NO:38HStructural domain and the V as shown in SEQ ID NO:40 or SEQ ID NO:39LStructure Domain.
8. antibody described in any one of -7 according to claim 1, the antibody is recombinant.
9. antibody described in any one of -7 according to claim 1, the antibody is Fab', F (ab') 2, F (ab') 3, unit price ScFv, bivalent scFv or single domain antibody.
10. antibody according to claim 1, the antibody is human antibody, humanized antibody or goes immune antiboidy.
11. antibody according to claim 1, the antibody conjugate is in preparation, chemotherapeutant, toxin or radioactive nucleus Element.
12. a kind of composition, it includes in pharmaceutically acceptable carrier according to claim 1 described in any one of -11 Antibody.
13. a kind of isolated polynucleotide molecule, it includes the cores for encoding antibody described in any one of -11 according to claim 1 Acid sequence.
14. a kind of method for manufacturing antibody comprising:
(a) one or more V for encoding antibody described in any one of -11 according to claim 1 are expressed in cellHAnd VLChain Polynucleotide molecule；With
(b) from antibody described in the cell purification.
15. a kind of for treating the composition of patient, wherein comprising a effective amount of according to claim 1 described in any one of -11 Antibody.
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